Overexpression of phytase genes in yeast systems

ABSTRACT

The present invention relates to a method of producing a heterologous protein or polypeptide having phytase activity in a yeast system. The invention also provides proteins having phytase activity which have increased thermostability. Yeast strains which produce a heterologous phytase and the vectors used to produce the phytase are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 11/962,446 filed Dec. 21, 2007, which is acontinuation application of U.S. patent application Ser. No. 11/372,851filed Mar. 10, 2006 and issued as U.S. Pat. No. 7,312,063 on Dec. 25,2007, which is a continuation application of U.S. patent applicationSer. No. 10/094,693 filed Mar. 8, 2002 and issued as U.S. Pat. No.7,026,150 on Apr. 11, 2006, which is a continuation-in-part of U.S.patent application Ser. No. 09/104,769 filed Jun. 25, 1998 and issued asU.S. Pat. No. 6,451,572 on Sep. 17, 2002, the contents of each of whichare incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to a method of producing phytase in yeast,yeast strains which express heterologous phytase, and the heterologousphytase produced by yeast.

BACKGROUND OF THE INVENTION

Phytases, a specific group of monoester phosphatases, are required toinitiate the release of phosphate (“P”) from phytate (myo-inositolhexophosphate), the major storage form of P in cereal foods or feeds(Reddy, N. R. et al., “Phytates in Legumes and Cereals,” Advances inFood Research, 28:1 (1982)). Because simple-stomached animals like swineand poultry as well as humans have little phytase activity in theirgastrointestinal tracts, nearly all of the ingested phytate P isindigestible. This results in the need for supplementation of inorganicP, an expensive and non-renewable nutrient, in diets for these animals.More undesirably, the unutilized phytate-P excreted through manure ofthese animals becomes P pollution of the environment (Cromwell, G. L. etal., “P—A Key Essential Nutrient, Yet a Possible Major Pollutant—ItsCentral Role in Animal Nutrition,” Biotechnology In the Feed Industry;Proceedings Alltech 7th Annual Symposium, p. 133 (1991)). Furthermore,phytate chelates with essential trace elements like zinc and producesnutrient deficiencies such as growth and mental retardation in childreningesting mainly plant origin foods without removal of phytate.

Two phytases, phyA and phyB, from Aspergillus niger NRRL3135 have beencloned and sequenced (Ehrlich, K. C. et al., “Identification and Cloningof a Second Phytase Gene (phys) from Aspergillus niger (ficuum),”Biochem. Biophys. Res. Commun., 195:53-57 (1993); Piddington, C. S. etal., “The Cloning and Sequencing of the Genes Encoding Phytase (phy) andpH 2.5-optimum Acid Phosphatase (aph) from Aspergillus niger var.awamori,” Gene, 133:56-62 (1993)). Recently, new phytase genes have beenisolated from Aspergillus terreus and Myceliophthora thermophila(Mitchell et al., “The Phytase Subfamily of Histidine Acid Phosphatases:Isolation of Genes for Two Novel Phytases From the Fungi Aspergillusterreus and Myceliophthora thermophila,” Microbiology 143:245-252,(1997)), Aspergillus fumigatus (Pasamontes et al., “Gene Cloning,Purification, and Characterization of a Heat-Stable Phytase from theFungus Aspergillus fumigatus” Appl. Environ. Microbiol., 63:1696-1700(1997)), Emericella nidulans and Talaromyces thermophilus (Pasamontes etal., “Cloning of the Phytase from Emericella nidulans and theThermophilic Fungus Talaromyces thermophilus,” Biochim. Biophys. Acta.,1353:217-223 (1997)), and maize (Maugenest et al., “Cloning andCharacterization of a cDNA Encoding a Maize Seedling Phytase,” Biochem.J. 322:511-517, 1997)).

Various types of phytase enzymes have been isolated and/or purified fromEnterobacter sp. 4 (Yoon et al., “Isolation and Identification ofPhytase-Producing Bacterium, Enterobacter sp. 4, and EnzymaticProperties of Phytase Enzyme,” Enzyme and Microbial Technology18:449-454 (1996)), Klebsiella terrigena (Greiner et al., “Purificationand Characterization of a Phytase from Klebsiella terrigena.,” Arch.Biochem. Biophys. 341:201-206 (1997)), and Bacillus sp. DS11 (Kim etal., “Purification and Properties of a Thermostable Phytase fromBacillus sp. DS11,” Enzyme and Microbial Technology 22:2-7 (1998)).Properties of these enzyme have been studied. In addition, the crystalstructure of phy A from Aspergillus ficuum has been reported (Kostrewaet al., “Crystal Structure of Phytase from Aspergillus ficuum at 2.5 AResolution,” Nature Structure Biology 4:185-190 (1997)).

Hartingsveldt et al. introduced phyA gene into A. niger and obtained aten-fold increase of phytase activity compared to the wild type.(“Cloning, Characterization and Overexpression of the Phytase-EncodingGene (phyA) of Aspergillus Niger,” Gene 127:87-94 (1993)). Supplementalmicrobial phytase of this source in the diets for pigs and poultry hasbeen shown to be effective in improving utilization of phytate-P andzinc (Simons et al., “Improvement of Phosphorus Availability ByMicrobial Phytase in Broilers and Pigs,” Br. J. Nutr., 64:525 (1990);Lei, X. G. et al., “Supplementing Corn-Soybean Meal Diets With MicrobialPhytase Linearly Improves Phytate P Utilization by Weaning Pigs,” J.Anim. Sci., 71:3359 (1993); Lei, X. G. et al., “SupplementingCorn-Soybean Meal Diets With Microbial Phytase Maximizes Phytate PUtilization by Weaning Pigs,” J. Anim. Sci., 71:3368 (1993); Cromwell,G. L. et al., “P—A Key Essential Nutrient, Yet a Possible MajorPollutant—Its Central Role in Animal Nutrition,” Biotechnology In theFeed Industry; Proceedings Alltech 7th Annual Symposium, p. 133 (1991)).But, expenses of the limited available commercial phytase supply and theactivity instability of the enzyme to heat of feed pelleting precludeits practical use in animal industry (Jongbloed, A. W. et al., “Effectof Pelleting Mixed Feeds on Phytase Activity and Apparent Absorbabilityof Phosphorus and Calcium in Pigs,” Animal Feed Science and Technology,28:233-242 (1990)). Moreover, phytase produced from A. niger ispresumably not the safest source for human food manufacturing.

Yeast can be used to produce enzymes effectively while grown on simpleand inexpensive media. With a proper signal sequence, the enzyme can besecreted into the media for convenient collection. Some yeast expressionsystems have the added advantage of being well accepted in the foodindustry and are safe and effective producers of food products.

Pichia pastoris is a methylotrophic yeast, capable of metabolizingmethanol as its sole carbon source. This system is well-known for itsability to express high levels of heterologous proteins. Because it isan eukaryote, Pichia has many of the advantages of higher eukaryoticexpression systems such as protein processing, folding, andpost-transcriptional modification.

Thus, there is a need to develop an efficient and simple system toproduce phytase economically for the application of food and feedindustry.

SUMMARY OF THE INVENTION

The present invention relates to a method of producing phytase in yeastby introducing a heterologous gene which encodes a protein orpolypeptide with phytase/acid phosphatase activity into a yeast strainand expressing that gene.

The present invention also relates to a protein or polypeptide havingphytase activity with optimum activity in a temperature range of 57-65°C. at pH of 2.5 to 3.5 or of 5.5. Optimal pH at 2.5 to 3.5 isparticularly important for phytase, because that is the stomach pH ofanimals.

The invention further provides a yeast cell carrying a heterologous genewhich encodes a protein or polypeptide with phytase activity and whichis functionally linked to a promoter capable of expressing phytase inyeast.

Yet another aspect of the invention is a vector having a gene from anon-yeast organism which encodes a protein or polypeptide with phytaseactivity, a promoter which is capable of initiating transcription inyeast functionally linked to the gene encoding a peptide with phytaseactivity, and with an origin of replication capable of maintaining thevector in yeast or being capable of integrating into the host genome.

The invention also provides a method for producing a protein orpolypeptide having phytase activity. An isolated appA gene, whichencodes a protein or polypeptide with phytase activity, is expressed ina host cell.

The invention also includes a method of converting phytate to inositoland inorganic phosphate. The appA gene expresses a protein ofpolypeptide with phytase activity in a host cell. The protein orpolypeptide is then contacted with phytate to catalyze the conversion ofphytate to inositol and inorganic phosphate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an SDS-PAGE analysis of soluble protein prepared from thephytase gene transformed E. coli induced with IPTG. The cells were grown4 hours before harvesting. Lane 1: Marker; Lanes 2 and 3: Transformantsof pEP1 (the expressed protein was approximately 55 kDa); Lane 4:Transformant with only the expression vector pET25b(+).

FIG. 2 shows the Western blot analysis of the expressed phytase proteinin E. coli. The antibody was raised against purified native phytase ofA. niger. Each lane contained 50 μg total intracellular protein. Lanes 1and 2: Recombinants after and before induction. Lanes 3 and 4: control(only the expressing vector) after and before induction.

FIG. 3 is a scan image of Northern blotting analysis of the mRNA of PhyAin E. coli. A 1.4 kb PhyA probe was used. Each lane contained 20 μg oftotal RNA. Lanes 1 and 2: RNA isolated from the control cells (only theexpression vector) before and after induction. Lanes 3 and 4: RNAisolated from the recombinants containing PhyA before and afterinduction.

FIG. 4 is a time course of the induced expression of phytase (pEP1) inE. coli BL21(DE3). The cells were induced when the OD₆₀₀ reached 0.5.The soluble protein, prepared at each time point, was quantified bySDS-PAGE analysis.

FIG. 5 shows an SDS-PAGE analysis of the expressed extracellular phytaseprotein by the phytase transformed S. lividans after growing for 72hours. Cells were spun for 15 minutes at 8,000×g, and the supernatantwas subjected to gel electrophoresis. Lane 1: Marker; Lane 2: Controlwith the only expression vector; Lane 3: Positive colony expressedphytase and the size was approximately 57 kDa.

FIG. 6 shows the Western blot analysis of the phytase expressed by S.lividans, using a phytase antibody raised against purified nativephytase of A. niger. Each lane was loaded with 20 μg medium(supernatant) protein. Lane 1: Supernatant from the vector transformedcontrol cell culture; Lane 2: Supernatant from the culture inoculatedwith the positive colony.

FIG. 7 depicts an SDS-PAGE analysis of the extracellular phytaseexpressed by S. cerevisiae. Each lane was loaded with 50 μg medium(supernatant) protein. Lanes 1 to 3: Supernatant from the cultureinoculated with the positive colony harvested at 5, 10, and 25 hoursafter induction, respectively; Lanes 4 to 6: Supernatant from thevector-transformed control cell culture harvested at 5, 10, and 25 hoursafter induction, respectively; Lane 7: Marker (kDa). The expressedphytase was approximately 110 kDa (confirmed by Western blot).

FIG. 8 is a time course of the extracellular phytase activity expressedby the pYPP1 construct transformed S. cerevisiae after induced bygalactose. The activity was analyzed in the supernatant of the collectedmedium.

FIG. 9 shows the Western blot analysis of the extracellular phytaseexpressed by S. cerevisiae before and after deglycosylation (Endo H),using a phytase antibody raised against purified native phytase in A.niger. Lane 1: Prestained SDS-PAGE standards (kDa) from Bio-Rad; Lanes 2and 3: deglycosylated 10 and 20 μg phytase protein, respectively; Lane4: glycosylated phytase (20 μg protein).

FIG. 10 is a scan image of Northern blot analysis for total RNA isolatedfrom transformed S. cerevisiae cells. Lane 1: Control (with only theexpression vector pYES2); Lanes 2 and 3: Transformants of pYPP1.

FIG. 11 is a time course of the extracellular phyA phytase activityproduced by Pichia pastoris transformants of Mut^(s) (KM71) and Mut+(X33) after induction.

FIG. 12 depicts an SDS-PAGE analysis of the overexpressed phytase inPichia, with construct of pPICZαA-PhyA in KM71 (MUT^(s)). Lane 1:protein ladder. Lane 2: 40 μl of the supernatant of AK1 (a colony showed21,700 mU/ml of extracellular phytase), collected 108 hours afterinduction. Lane 3: 40 μl of the supernatant of a control strainoverexpressing human serum albumin (HAS, 6.7 kDa) at a level of 1 g/L.Lane 4: 40 μl of the supernatant of the KM71 control.

FIG. 13 depicts effects of deglycosylation by Endo H on thethermostability of the expressed phytase in Pichia. Phytase activity wasmeasured after the enzymes were heated for 15 minutes under 37 or 80° C.in 0.2 M citrate buffer, pH 5.5.

FIG. 14 is a scan image of Northern analysis of the expressed phyA mRNAby the transformed Pichia pastoris strains (KM71 and X33). A 1.3 kb phyAprobe was used for blotting. Lanes 1 and 2: the transformant of KM71before and after induction; Lanes 3 and 4: the transformant of X33 afterand before induction.

FIG. 15 shows the optimum pH of the expressed extracellular phytase byPichia (X33). Buffers of 0.2 M glycine-HCl for pH 1.5, 2.5, 3.5; 0.2 Msodium citrate for pH 4.5, 5.5, 6.5, and 0.2 M Tris-HCl for pH 7.5 and8.5 were used.

FIG. 16 shows the optimum temperature of the expressed extracellularphytase by Pichia (X33). The assays were conducted in 0.2 M citratebuffer, pH 5.5.

FIG. 17 depicts the release of free phosphorus from soybean by theexpressed phytase in Pichia (X33). Five grams of soybean were suspendedin 25 ml of 0.2 M citrate, pH 5.5, with different amounts of the enzyme.The incubation was conducted for 4 hours under 37° C. and the freephosphorus released in the supernatant was determined.

FIG. 18 shows a time course of the expression of the extracellularphytase activity from five transformants of Pichia pastoris containingthe E. coli appA gene.

FIG. 19 graphically shows the relationship between medium pH and theexpression of phytase activity by Pichia pastoris.

FIG. 20 is an SDS-PAGE analysis of the E. coli phytase overexpressed inPichia pastoris. Lane 1: Protein ladder; Lanes 2 to 4: Supernatantscollected from the cultures of positive colonies 23, 22, and 11,respectively, at 118 hours after induction.

FIG. 21 graphically shows the optimum pH of the overexpressed E. coliphytase by Pichia pastoris.

FIG. 22 graphically shows the optimum temperature of the overexpressedE. coli phytase by Pichia pastoris.

FIG. 23 shows the amount of free phosphorus released from soybean mealby the overexpressed E. coli phytase from Pichia pastoris after fourhours treatment.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of producing phytase in yeast.According to this method, a heterologous gene which encodes a protein orpolypeptide with phytase activity is expressed in a yeast strain.

The enzymes which catalyze the conversion of phytate to inositol andinorganic phosphorus are broadly known as phytases. Phytase producingmicroorganisms comprise bacteria such as Bacillus subtilis (Paver etal., J. Bacteriol. 151, 1102 (1982), which is hereby incorporated byreference) and Pseudomonas (Cosgrove, Austral. J. Biol. Sci. 23:1207(1970), which is hereby incorporated by reference); yeasts, such asSaccharomyces cerevisiae (Nayini et al., Lebensmittel Wissenschaft andTechnologie 17:24 (1984), which is hereby incorporated by reference);and fungi, such as Aspergillus terreus (Yamada et al., Agric. Biol.Chem. 32:1275 (1986), which is hereby incorporated by reference), andAspergillus ficuum (van Gorcom et al., European Patent Application89/202,436, which is hereby incorporated by reference).

Phytases are also endogenously present in many plant species. Loewus,In: Plant Biology vol. 9: “Inositol Metabolism in Plants” (eds. D. J.Morre, W. F. Boss, F. A. Loewus) 13 (1990); and Gellatly, et al., PlantPhysiology (supplement) 93:562 (1990), which are hereby incorporated byreference, mention the isolation and characterization of a phytase cDNAclone obtained from potato tubers. Gibson, et al., J. Cell Biochem.,12C:L407 (1988) and Christen, et al., J. Cell Biochem., 12C:L402 (1988),which are hereby incorporated by reference, mentions the synthesis ofendogenous phytase during the germination of soybean seeds.

Preferably, the protein or polypeptide with phytase activity is secretedby the cell into growth media. This allows for higher expression levelsand easier isolation of the product. The protein or polypeptide withphytase activity is coupled to a signal sequence capable of directingthe protein out of the cell. Preferably, the signal sequence is cleavedfrom the protein.

In a preferred embodiment, the heterologous gene, which encodes aprotein or polypeptide with phytase activity, is spliced in frame with atranscriptional enhancer element.

Preferred heterologous genes encoding proteins with phytase activity areisolated from a bacterial cell. A more preferred gene is the phyA geneof Aspergillus niger. A gene encoding phytase, phyA, from Aspergillusniger NRRL3135 has been cloned and sequenced (Piddington, C. S. et al.,“The Cloning and Sequencing of the Genes Encoding Phytase (phy) and pH2.5-optimum Acid Phosphatase (aph) from Aspergillus niger var. awamori,”Gene, 133:56-62 (1993), which are hereby incorporated by reference).Hartingsveldt et al. introduced phyA gene into A. niger, and obtained atenfold increase of phytase activity compared to the wild type.(Hartingsveldt et al., “Cloning, Characterization and Overexpression ofthe Phytase-Encoding Gene (phyA) of Aspergillus Niger,” Gene 127:87-94(1993), which is hereby incorporated by reference.)

Another preferred heterologous gene is the appA gene of E. coli. Thegene, originally defined as E. coli periplasmic phosphoanhydridephosphohydrolase (appA) gene, contains 1,298 nucleotides (GeneBankaccession number: M58708). The gene was first found to code for an acidphosphatase protein of optimal pH of 2.5 (EcAP) in E. coli. The acidphosphatase is a monomer with a molecular mass of 44,644 daltons. MatureEcAP contains 410 amino acids (Dassa, J. et al., “The CompleteNucleotide Sequence of the Escherichia Coli Gene AppA RevealsSignificant Homology Between Ph 2.5 Acid Phosphatase andGlucose-1-Phosphatase,” J. Bacteriology, 172:5497-5500 (1990), which ishereby incorporated by reference). Ostanin, et al. overexpressed appA inE. coli BL21 using a pT7 vector and increased its acid phosphataseactivity by approximately 400-folds (440 mU/mg protein) (Ostanin, K. etal., “Overexpression, Site-Directed Mutagenesis, and Mechanism ofEscherichia Coli Acid Phosphatase,” J. Biol. Chem., 267:22830-36 (1992),which is hereby incorporated by reference). The product of the appA genewas not previously known to have phytase activity.

The appA or phyA gene can be expressed in any prokaryotic or eukaryoticexpression system. A variety of host-vector systems may be utilized toexpress the protein-encoding sequence(s). Preferred vectors include aviral vector, plasmid, cosmid or an oligonucleotide. Primarily, thevector system must be compatible with the host cell used. Host-vectorsystems include but are not limited to the following: bacteriatransformed with bacteriophage DNA, plasmid DNA, or cosmid DNA;microorganisms such as yeast containing yeast vectors; mammalian cellsystems infected with virus (e.g., vaccinia virus, adenovirus, etc.);insect cell systems infected with virus (e.g., baculovirus); and plantcells infected by bacteria. The expression elements of these vectorsvary in their strength and specificities. Depending upon the host-vectorsystem utilized, any one of a number of suitable transcription andtranslation elements can be used.

Preferred hosts for expressing appA or phyA include fungal cells,including species of yeast or filamentous fungi, may be used as hostcells in accordance with the present invention. Preferred yeast hostcells include different strains of Saccharomyces cerevisiae. Otheryeasts like Kluyveromyces, Torulaspora, and Schizosaccharomyces can alsobe used. In a preferred embodiment, the yeast strain used to overexpressthe protein is Saccharomyces cerevisiae. Preferred filamentous fungihost cells include Aspergillus and Neurospora. A more preferred strainof Aspergillus is Aspergillus niger.

In another preferred embodiment of the present invention, the yeaststrain is a methylotrophic yeast strain. Methylotrophic yeast are thoseyeast genera capable of utilizing methanol as a carbon source for theproduction of the energy resources necessary to maintain cellularfunction and containing a gene for the expression of alcohol oxidase.Typical methylotrophic yeasts include members of the genera Pichia,Hansenula, Torulopsis, Candida, and Karwinskia. These yeast genera canuse methanol as a sole carbon source. In a more preferred embodiment,the methylotrophic yeast strain is Pichia pastoris.

The present invention also provides a protein or polypeptide withphytase activity. PhyA is expressed in Pichia and the resulting proteinproduced has much higher extracellular activity (˜65 mU/ml). The phytaseactivity yield was approximately 30-fold greater than that in phyAtransformed Saccharomyces cerevisiae, 21-fold greater than that in wildtype of Aspergillus niger, and 65,000-fold greater than that in theuntransformed Pichia. The optimal pH of the expressed phytase was 2.5and 5.5, and the optimal temperature was 60° C. Similarly, appA isexpressed in Pichia and Saccharomyces cerevisiae with the resultingprotein having much higher extracellular activity and a much preferredoptimal pH of 2.5 to 3.5.

A preferred embodiment of the invention is a protein or polypeptidehaving phytase activity with optimum activity in a temperature range of57 to 65° C. A more preferred embodiment is a protein or polypeptidehaving phytase activity, where its temperature range for optimumactivity is from 58 to 62° C.

Yet another preferred embodiment is a protein or polypeptide havingphytase activity where the protein retains at least 40% of its activityafter heating the protein for 15 minutes at 80° C. More preferred is aprotein or polypeptide having phytase activity where the protein retainsat least 60% of its activity after heating the protein for 15 minutes at60° C.

Purified protein may be obtained by several methods. The protein orpolypeptide of the present invention is preferably produced in purifiedform (preferably at least about 80%, more preferably 90%, pure) byconventional techniques. Typically, the protein or polypeptide of thepresent invention is secreted into the growth medium of recombinant hostcells. Alternatively, the protein or polypeptide of the presentinvention is produced but not secreted into growth medium. In suchcases, to isolate the protein, the host cell carrying a recombinantplasmid is propagated, lysed by sonication, heat, or chemical treatment,and the homogenate is centrifuged to remove cell debris. The supernatantis then subjected to sequential ammonium sulfate precipitation. Thefraction containing the polypeptide or protein of the present inventionis subjected to gel filtration in an appropriately sized dextran orpolyacrylamide column to separate the proteins. If necessary, theprotein fraction may be further purified by HPLC.

The present invention also provides a yeast strain having a heterologousgene which encodes a protein or polypeptide with phytase activity. Theheterologous gene should be functionally linked to a promoter capable ofexpressing phytase in yeast.

Yet another aspect of the invention is a vector for expressing phytasein yeast. The vector carries a gene from a non-yeast organism whichencodes a protein or polypeptide with phytase activity. The phytase genecan be cloned into any vector which replicates autonomously orintegrates into the genome of yeast. The copy number of autonomouslyreplicating plasmids, e.g. YEp plasmids may be high, but their mitoticstability may be insufficient (Bitter et al., “Expression and SecretionVectors for Yeast,” Meth. Enzymol. 153:516-44 (1987), which is herebyincorporated by reference). They may contain the 2 mu-plasmid sequenceresponsible for autonomous replication, and an E. coli sequenceresponsible for replication in E. coli. The vectors preferably contain agenetic marker for selection of yeast transformants, and an antibioticresistance gene for selection in E. coli. The episomal vectorscontaining the ARS and CEN sequences occur as a single copy per cell,and they are more stable than the YEp vectors. Integrative vectors areused when a DNA fragment is integrated as one or multiple copies intothe yeast genome. In this case, the recombinant DNA is stable and noselection is needed (Struhl et al., “High-Frequency Transformation ofYeast: Autonomous Replication of Hybrid DNA Molecules,” Proc. Nat'lAcad. Sci. USA 76:1035-39 (1979); Powels et al., Cloning Vectors, I-IV,et seq. Elsevier, (1985); Sakai et al., “Enhanced Secretion of HumanNerve Growth Factor from Saccharomyces Cerevisiae Using an Advancedδ-Integration System,” Biotechnology 9:1382-85 (1991), which are herebyincorporated by reference). Some vectors have an origin of replication,which functions in the selected host cell. Suitable origins ofreplication include 2μ, ARS1, and 25 μM. The vectors have restrictionendonuclease sites for insertion of the fusion gene and promotersequences, and selection markers. The vectors may be modified by removalor addition of restriction sites, or removal of other unwantednucleotides.

The phytase gene can be placed under the control of any promoter(Stetler et al., “Secretion of Active, Full- and Half-Length HumanSecretory Leukocyte Protease Inhibitor by Saccharomyces cerevisiae,”Biotechnology 7:55-60, (1989), which is hereby incorporated byreference). One can choose a constitutive or regulated yeast promoter.Suitable promoter sequences for yeast vectors include, among others,promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman etal., J. Biol. Chem. 255:2073 (1980), which is hereby incorporated byreference) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg.7:149 (1968); and Holland et al., Biochem. 17:4900, (1978), which arehereby incorporated by reference), such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. Other suitable vectors andpromoters for use in yeast expression are further described in EPA-73,657 to Hitzeman, which is hereby incorporated by reference. Anotheralternative is the glucose-repressible ADH2 promoter described byRussell et al., J. Biol. Chem. 258:2674 (1982) and Beier et al., Nature300:724 (1982), which are hereby incorporated by reference.

One can choose a constitutive or regulated yeast promoter. The strongpromoters of e.g., phosphoglycerate kinase (PGK) gene, other genesencoding glycolytic enzymes, and the alpha-factor gene, areconstitutive. When a constitutive promoter is used, the product issynthesized during cell growth. The ADH2 promoter is regulated withethanol and glucose, the GAL-1-10 and GAL7 promoters with galactose andglucose, the PHO5 promoter with phosphate, and the metallothioninepromoter with copper. The heat shock promoters, to which the HSP150promoter belongs, are regulated by temperature. Hybrid promoters canalso be used. A regulated promoter is used when continuous expression ofthe desired product is harmful for the host cells. Instead of yeastpromoters, a strong prokaryotic promoter such as the T7 promoter, can beused, but in this case the yeast strain has to be transformed with agene encoding the respective polymerase. For transcription termination,the HSP150 terminator, or any other functional terminator is used. Here,promoters and terminators are called control elements. The presentinvention is not restricted to any specific vector, promoter, orterminator.

The vector may also carry a selectable marker. Selectable markers areoften antibiotic resistance genes or genes capable of complementingstrains of yeast having well characterized metabolic deficiencies, suchas tryptophan or histidine deficient mutants. Preferred selectablemarkers include URA3, LEU2, HIS3, TRP1, HIS4, ARG4, or antibioticresistance genes.

The vector may also have an origin of replication capable of replicationin a bacterial cell. Manipulation of vectors is more efficient inbacterial strains. Preferred bacterial origin of replications are ColE1,Ori, or oriT.

A leader sequence either from the yeast or from phytase genes or othersources can be used to support the secretion of expressed phytase enzymeinto the medium. The present invention is not restricted to any specifictype of leader sequence or signal peptide.

Suitable leader sequences include the yeast alpha factor leadersequence, which may be employed to direct secretion of the phytase. Thealpha factor leader sequence is often inserted between the promotersequence and the structural gene sequence (Kurjan et al., Cell 30:933,(1982); Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330, (1984); U.S.Pat. No. 4,546,082; and European published patent application No.324,274, which are hereby incorporated by reference). Another suitableleader sequence is the S. cerevisiae MF alpha 1 (alpha-factor) issynthesized as a prepro form of 165 amino acids comprising signal- orprepeptide of 19 amino acids followed by a “leader” or propeptide of 64amino acids, encompassing three N-linked glycosylation sites followed by(LysArg(Asp/Glu, Ala)2-3 alpha-factor)4 (Kurjan, et al., Cell 30:933-43(1982), which is hereby incorporated by reference). The signal-leaderpart of the preproMF alpha 1 has been widely employed to obtainsynthesis and secretion of heterologous proteins in S. cerivisiae. Useof signal/leader peptides homologous to yeast is known from. U.S. Pat.No. 4,546,082, European Patent Applications Nos. 116,201; 123,294;123,544; 163,529; and 123,289 and DK Patent Application No. 3614/83,which are hereby incorporated by reference. In European PatentApplication No. 123,289, which is hereby incorporated by reference,utilization of the S. cerevisiae a-factor precursor is described whereasWO 84/01153, which is hereby incorporated by reference, indicatesutilization of the Saccharomyces cerevisiae invertase signal peptide,and German Patent Application DK 3614/83, which is hereby incorporatedby reference, indicates utilization of the Saccharomyces cerevisiae PH05signal peptide for secretion of foreign proteins.

The alpha-factor signal-leader from Saccharomyces cerevisiae (MF alpha 1or MF alpha 2) may also be utilized in the secretion process ofexpressed heterologous proteins in yeast (U.S. Pat. No. 4,546,082,European Patent Applications Nos. 16,201; 123,294; 123 544; and 163,529,which are hereby incorporated by reference). By fusing a DNA sequenceencoding the S. cerevisiea MF alpha 1 signal/leader sequence at the 5′end of the gene for the desired protein secretion and processing of thedesired protein was demonstrated. The use of the mouse salivary amylasesignal peptide (or a mutant thereof) to provide secretion ofheterologous proteins expressed in yeast has been described in PublishedPCT Applications Nos. WO 89/02463 and WO 90/10075, which are herebyincorporated by reference.

U.S. Pat. No. 5,726,038 describes the use of the signal peptide of theyeast aspartic protease 3, which is capable of providing improvedsecretion of proteins expressed in yeast. Other leader sequencessuitable for facilitating secretion of recombinant polypeptides fromyeast hosts are known to those of skill in the art. A leader sequencemay be modified near its 3′ end to contain one or more restrictionsites. This will facilitate fusion of the leader sequence to thestructural gene.

Yeast transformation protocols are known to those of skill in the art.One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci.USA 75:1929 (1978), which is hereby incorporated by reference. TheHinnen et al. protocol selects for Trp transformants in a selectivemedium, wherein the selective medium consists of 0.67% yeast nitrogenbase, 0.5% casamino acids, 2% glucose, 10 μg/ml adenine and 20 μg/mluracil.

The gene may be maintained on stable expression vector, an artificialchromosome, or by integration into the yeast host cell chromosome.Integration into the chromosome may be accomplished by cloning thephytase gene into a vector which will recombine into a yeast chromosome.Suitable vectors may include nucleotide sequences which are homologousto nucleotide sequences in the yeast chromosome. Alternatively, thephytase gene may be located between recombination sites, such astransposable elements, which can mobilize the gene into the chromosome.

The present invention also provides a method of producing phytase byproviding an isolated appA gene, which encodes a protein or polypeptidewith phytase activity, and expressing the gene in host cell. Preferablythe appA gene is isolated from Escherichia coli. Preferred host cellsinclude yeast or filamentous fungi. The preferred filamentous fungi isAspergillus niger and the preferred yeast are Saccharomyces,Kluyveromyces, Torulaspora, and Schizosaccharomyces, in particular, theyeast strain, Saccromyces cerivesia.

A method of converting phytate to inositol and inorganic phosphorus isalso provided. An appA gene is isolated from an organism, usingtechniques well known in the art. A protein or polypeptide with phytaseactivity is then expressed from the gene in a host cell. The resultingprotein or polypeptide is mixed or contacted with phyate. This techniqueis especially useful for treating phytate in food or animal feed.

The preferred appA gene is isolated from Escherichia coli.

While the phytase enzyme produced in a yeast system released phytate-Pfrom corn and soy as effectively as the currently commercial phytase, itappeared to be more thermostable. This phytase overexpression system inyeast can be used to provide thermostable phytase for use in the foodand feed industries.

EXAMPLES Example 1 Materials and Methods for Overexpressing PhyA in E.Coli, S. Lividans, and a Saccharomyces System

Phytase gene, host strains, and expression plasmids. Phytase gene, phyA,was kindly provided by Dr. E. J. Mullaney of the USDA. The gene (GenBankAccession number M94550) was contained in plasmid pMD4.21 in E. colistrain HB101. A 2.7 kb SphI fragment of A. niger DNA contained thecoding region of the deglycosylated phytase and its 5′ and 3′ flankingsequences. A plasmid containing the signal peptide sequence, Spxy, ofthe active xylanase gene of Aureobasidum pullulans (GenBank Accessionnumber U10298) was kindly provided by Dr. X. L. Li of the University ofGeorgia. The E. coli strain DH5α was used as an initial host for all therecombinant plasmids. In order to express phyA in E. coli, theexpression vector, pET25b(+) (Novagen, Madison, Wis.) and the expressionhost, BL21 (DE3)pLysS, were used. In order to express phyA in S.lividans TK 24, plasmid pSES1 (Jung, E. D. et al., “DNA Sequences andExpression in Streptomyces Lividansoglucanase Gene and an EndoglucanaseGene from Thermomonospora Fusca,” Appl. Environ. Microbiol., 59:3032-43(1993), which is hereby incorporated by reference), was used toconstruct the shuttle plasmid (from Dr. D. B. Wilson of CornellUniversity and he obtained it from Dr. D. A. Hopwood, John InnesInstitute, Norwich, England). In order to express phyA in yeast, theexpression vector pYES2 and the host S. cerevisiae strain, INVSc1(Invitrogen, San Diego, Calif.) were used.

Plasmid cassette constructions and transformations. All the constructedplasmids and the correspondent hosts are listed in Table 1. A 1.4 kb PCRfragment of phyA gene was amplified from the pMD4.21 by using twoprimers: upstream 5′-CGG AAT TCG TCA CCT CCG GAC T-3′ (SEQ ID No. 1) anddownstream 5′-CCC AAG CTT CTA AGC AAA ACA CTC-3′ (SEQ ID No. 2). Theresulting fragment contained the sequence coding for the deglycosylatedphytase of A. niger, PhyA, and EcoRI and HindIII restriction siteupstream and downstream, respectively. After purification with GenecleanII kit (Bio101, Inc., La Jolla, Calif.), the fragment was inserted intopET25b(+), and the resulting construct pEP1 (6893 bp) was transformedinto BL21(DE3)pLysS after initial confirmation in DH5α cells. Theexpression was under the control of T7 promoter followed by the leadsequence (pel B) encoding 21 amino acids, and phyA. The host transformedwith the pET25(+) vector only was used as the control.

TABLE 1 Expression vectors, constructs, and their host strains used inthe study Plasmid Host Description¹ Reference² pET25b(+) E. coli DH5αand Expression vector Novagen BL21 (DE3)pLysS pEP1 E. coli BL21pET25bα(+) + This paper (DE3)pLysS phyA gene PSES2 E. coli DH5α andExpression vector Jung et al.², S. lividans TK24 1993 PSPP1 E. coli DH5αand pSES2 + Spe2 + This paper S. lividans TK24 phyA PYES2 E. coli DH5αand Expression vector Invitrogen S. cerevisiae INVSc1 PYEP1 E. coli DH5αand pYES2 + Spe2 + This paper S. cerevisiae INVSc1 phyA PYXP1 E. coliDH5α and pYES2 + Spxy + This paper S. cerevisiae INVSc1 phyA PYPP1 E.coli DH5α and pYES2 + Sphy + This paper S. cerevisiae INVSc1 phyA ¹Spe2is the signal peptide for endoglucanase E2 of T. fusca (Wilson, D. B.,“Biochemistry and Genetics of Actinomycete Cellulases,” Crit. Rev.Biotechnol., 12: 45-63 (1992), which is hereby incorporated byreference); Spxy is the signal peptide forxylanase of A. pullulans (Liand Ljungdahl, “Cloning, Sequencing, and Regulation of a Xylanase Genefrom the Fungus Aureobasidium pullulans Y-2331-1,” Appl. Environ.Microbiol., 60: 3160-66 (1994); Li and Ljungdahl, “Expression ofAureobasidium pullulans xynA in, and Secretion of the Xylanase from,Saccharomyces cerevisiae,” Appl. Environ. Microbiol., 62: 209-13 (1996),which are hereby incorporated by reference); and Sphy is the signalpeptide for phyA of A. niger (Hartingsveldt et al., “Cloning,Characterization and Overexpression of the Phytase-Encoding Gene (phyA)of Aspergillus Niger,” Gene 127: 87-94 (1993), which is herebyincorporated by reference). ²Jung, E. D. et al., “DNA Sequences andExpression in Streptomyces Lividansoglucanase Gene and an EndoglucanaseGene from Thermomonospora Fusca,” Appl. Environ. Microbiol., 59: 3032-43(1993), which is hereby incorporated by reference).

The construction of the plasmid for phyA expression in S. lividansstarted with the synthesis of a fragment containing pLT1 promoter andSpe2 signal peptide (Lao, G. et al., “DNA Sequences of ThreeBeta-1,4-endoglucanase Genes From Thermomonospora Fusca,” J. Bacteriol.,173:3397-407 (1991), which is hereby incorporated by reference) by PCR.An upstream primer, 5′-CAG CTA TGA CCA TGA TTA CGC C-3′ (SEQ ID No. 3),and a downstream primer, 5′-CCT AGA ACG GGA ATT CAT TGG CCG CC-3′ (SEQID No. 4), contained PstI and EcoRI restriction sites, respectively. Thefragment was amplified from pBW2 (Jung, E. D. et al., “DNA Sequences andExpression in Streptomyces Lividans of an Exoglucanase Gene and anEndoglucanase Gene From Thermomonospora Fusca,” Appl. Environ.Microbiol., 59:3032-43 (1993), which is hereby incorporated byreference) and then digested with PstI and EcoRI, while the constructpEP1 and plasmid pBluescript SK⁺ (Strategene, La Jolla, Calif.) weredigested with EcoRI and HindIII, and PstI and HindIII, respectively. Thethree digested fragments were subsequently purified using Geneclean IIkit and ligated into a single recombinant construct that contained thedesired restriction sites of PstI and KpnI (from pBluescript SK*), pLT1promoter and Spe2 leading peptide of endoglucanase E2 (551 bp, Lao, G.et al., “DNA Sequences of Three Beta-1,4-endoglucanase Genes FromThermomonospora Fusca,” J. Bacteriol., 173:3397-407 (1991), which ishereby incorporated by reference), and phyA gene (1365 bp). After theconstruct was digested with PstI and KpnI, the resulting fragment wasinserted into the expression vector pSES1, and the formed shuttleplasmid (pSPP1, 9131 bp) was transformed into the host S. lividansprotoplasts according to Hopwood et al. (Hopwood, D. A., et al., GeneticManipulation of Streptomyces-A Laboratory Manual, The John InnesFoundation, Norwich, England (1985), which is hereby incorporated byreference). Likewise, a control was prepared by transforming S. lividanswith expression vector pSES2.

Three shuttle plasmids with three different signal peptide sequenceswere constructed to express phyA in the yeast system (See Table 2). Thefirst plasmid was originated from a HindIII digested fragment of pSPp1,including the promoter pLT1, lead sequence Spe2, and the coding regionsequence of phyA. The fragment was ligated into the HindIII site ofpYES2 treated with calf intestinal alkaline phosphatase and the plasmidwas named pYEP1 (7783 bp) after its right orientation was confirmed. Thesecond plasmid contained Spxy, a signal peptide sequence of xylanasegene from A. pullulans (Li, X. L. et al., “Cloning, Sequencing, andRegulation of a Xylanase Gene From the Fungus Aureobasidium Pullulans)Y-2311-1,” Appl. Environ. Microbiol., 60:3160-166 (1994); Li, X. L. etal., “Expression of Aureobasidium Pullulans XynA in, and Secretion ofthe Xylanase From, Saccharomyces Cerevisiaea,” Appl. Environ.Microbiol., 62:209-13 (1996), which are hereby incorporated byreference), and phyA gene. Spxy was spliced with phyA by overlapextension (Horton, R. M., “In Vitro Recombination and Mutagenesis ofDNA: SOEing Together Tailor-Made Genes,” PCR Protocols: Current Methodsand Applications, 251-61 (1993), which is hereby incorporated byreference) with two successive steps of PCR. One was to amplify Spxysequence from pCE4 (Li, X. L. et al., “Expression of AureobasidiumPullulans XynA in, and Secretion of the Xylanase From, SaccharomycesCerevisiaea,” Appl. Environ. Microbiol., 62:209-13 (1996), which ishereby incorporated by reference) using upstream primer (5′-CCC AAG CTTGAT CAC ATC CAT TCA-3′) (SEQ ID No. 5) with a HindIII restriction site(primer 1) and overlapping downstream primer (5′-CGG GGA CTG CTA GCG CACGTT CGA T-3′, primer 2) (SEQ ID No. 6). The other PCR was to amplify thecoding region of phyA from pEP1 using overlapping upstream primer(5′-ATC GAA CGT GCG CTA GCA GCA GTC CCC G-3′, primer 3) (SEQ ID No. 7)and downstream primer (5′-GCT CTA GAC TAA GCA AAA CAC TCC-3′, primer 4)(SEQ ID No. 8) with a XbaI restriction site. The second step of PCR wasconducted to merge the two fragments generated from the above two PCR byusing the two purified fragments as the templates and primers 1 and 4.The resulting fragment contained HindIII and XbaI restriction sites andwas cloned into pSES2. This plasmid was named pYXP1 (7219 bp). The thirdplasmid contained the signal peptide (Sphy) sequence of phyA and thecoding region of phyA, excluding the intron between them (Hartingsveldt,W. van., et al., “Cloning, Characterization and Overexpression of thePhytase-Encoding Gene (phyA) of Aspergillus Niger,” Gene 127:87-94(1993), which is hereby incorporated by reference). Two primers,including a 70 bp of upstream primer contained the signal peptide withan engineered KpnI restriction site and a downstream primer that was thesame one used for pYXP1 construction (primer 4) were used to amplify thedesired fragment from pEP1. The PCR product was digested with KpnI andXbaI and cloned into pSES2, resulting in a plasmid named pYPP1 (7176bp). All the above three constructs were transformed into S. cerevisiaeby the method of Ito et al., “Transformation of Intact Yeast CellsTreated with Alkali Cations,” J. Bacteriol., 153:163-68 (1983), which ishereby incorporated by reference.

TABLE 2 Signal peptides used for expression of phyA in S. cerevisiaeConstruct Phytase Size activity¹ (bp) Peptide Gene Organism (mPU/ml)pYEP1 7783 Spe2 Cellulase E2 T. fusca .80 (93 bp) pYXP1 7219 SpxyXylanase A A. pullulans Non-detectable (102 bp)  pYPP1 7176 Sphy PhyAPhytase A. niger 146 (57 bp) pSES1² 7224 S. cerevisiae Non-detectable¹The phytase activity was detected in the supernatant of cell culture ofSabouraud-raffinose medium 15 hours after induced by adding galactose.See text for definition of phytase units. ²Expression vector for S.cerevisiae, used as a control.

Growth medium and induction of the gene expression. In the E. colisystem, the transformants were grown in 50 ml of LB medium containing 50μg/ml of ampicillin at 30° C. After the OD₆₀₀ value of the mediumreached 0.5 to 0.6, phytase gene expression was induced by adding IPTG(isopropyl b-D-thiogalactopyranoside) into the medium to a finalconcentration of 1 mM. Three hours after the induction, cells werecollected by spinning down at 8000×g for 15 minutes, washed with 1×PBS,and lysed by lysozyme. Soluble and insoluble fractions of the cells wereprepared, and a sample containing 500 μg of total protein (Lowry, O. H.et al., “Protein Measurement With the Folin Phenol Reagent,” J. Biol.Chem., 193:265-75 (1951), which is hereby incorporated by reference) wassuspended in the same volume of 2×SDS buffer and analyzed by SDS-PAGE(Laemmli, U.K., “Cleavage of Structural Proteins During the Assembly ofthe Head of Bacteriophage T4,” Nature (London), 227:680-85 (1970), whichis hereby incorporated by reference).

Recombinant S. lividans was grown in TSB broth with 5 μg/ml ofthiostrepton at 30° C. (Jung, E. D. et al., “DNA Sequences andExpression in Streptomyces Lividans of an Exoglucanase Gene and anEndoglucanase Gene from Thermomonospora Fusca,” Appl. Environ.Microbiol., 59:3032-43 (1993), which is hereby incorporated byreference). After 72 hours incubation, the cells and medium wereharvested and prepared for SDS-PAGE (Wilson, D. B., “Biochemistry andGenetics of Actinomycete Cellulases,” Crit. Rev. Biotechnol., 12:45-63(1992), which is hereby incorporated by reference).

Transformants of S. cerevisiae were initially grown inSabouraud-raffinose (4%) medium (100 ml) without uracil for 48 hours,sterile galactose was then added into the medium (2%) to induce phytaseexpression. Samples of media and cells were collected at various timepoints, and extracellular and intracellular samples were prepared asdescribed by Li and Ljungdahl, “Expression of Aureobasidium pullulansxynA in, and Secretion of the Xylanase from, Saccharomyces cerevisiae,”Appl. Environ. Microbiol., 62:209-13 (1996), which is herebyincorporated by reference. When needed, the supernatant of the expressedcell culture fractions was concentrated with Stirred Cells of Amicon(Beverly, Mass.) by using YM10 membranes (MW cutoff of 10,000). Othermedia were tested accordingly.

Enzyme protein and activity assay. Amounts of expressed phytase proteinunder various conditions were quantified by the relative densitometry ofspecific bands in SDS-PAGE, using IS-1000 Digital Imaging System (AlphaInnotech Corporation, San Leandro, Calif.). Phytase activity in thesamples of media and cells was determined as previously described(Piddington, C. S. et al., “The Cloning and Sequencing of the GenesEncoding Phytase (phy) and pH 2.5-optimum Acid Phosphatase (aph) fromAspergillus niger var. awamori,” Gene, 133:56-62 (1993), which is herebyincorporated by reference) and the inorganic phosphate released wasassayed by the method of Chen, P. S. et al., “Microdetermination of P,”Anal. Chem., 28:1756-58 (1956), which is hereby incorporated byreference. One phytase unit (PU) was defined as the amount of enzymethat releases one μmol of inorganic phosphate from sodium phytate perminute at 37° C.

Western blotting (immunoblot) analysis. The soluble fraction of the cellmass of the phytase transformed E. coli and the medium supernatant of S.lividans and S. cerevisiae transformants were collected as for SDS-PAGE.After electrophoresis, the proteins were then transferred onto Protran®nitrocellulose membrane (Schleicher & Schuell, Keene, N.H., USA) in 20mM Tris-HCl (pH 8.3), 20% methanol, and 0.1% SDS, by using a MiniTrans-Blot cell (Bio-Rad Laboratories). Transfer was done overnight at aconstant 50 V and the initial buffer temperature was 4° C. The membraneswere then subjected to Western blot analysis. A rabbit polyclonal IgG(Kindly provided by Dr. A. H. J. Ullah of USDA. Dilution, 1: 5,000)against purified native A. niger phytase was used as the first antibody.The blotting was finalized using Immuno-Blot Assay Kit (Bio-RadLaboratories) containing a second antibody conjugated with horseradishperoxidase.

Total RNA isolation and analysis. Total RNA was isolated with TRIzol™Reagent (GIBCO BRL, Gaithersburg, Md.) from E. coli and S. cerevisiaetransformants 3 and 15 hours after induction, respectively. RNA samples(10 μg per lane) were then separated by formaldehyde agarose (1.5%,wt/vol) gel electrophoresis and transferred to Hyblot membranes(National Labnet, Woodbridge, N.J.) (Davis et al., Basic Methods inMolecular Biology, 2nd Ed., Appleton and Lange, Norwalle, Conn. (1994),which is hereby incorporated by reference). A 1.4 kb EcoRI-HindIIIfragment in plasmid pEP1 was prepared and was random-primed labeled with³²P using a DNA labeling kit followed by G-50 column purification(Pharmacia Biotech., Piscataway, N.J.) and then hybridized with theblotted RNA membranes in a hybridization oven (Hybaid, Middlesex, UK).The hybridized membranes were exposed to screens in Fuji Imaging Plateand analyzed by Bio-Imaging Analyzer (Kohshin Graphic Systems, Fuji,Japan).

Example 2 Expression of PhyA in E. coli

Four hours after the induction, a specific band (˜55 kDa) was viewed inSDS-PAGE (12.5%) of the soluble cell fraction, compared to the onlyexpression vector transformed control (See FIGS. 1 and 2). This bandrepresented 3.8% of the total soluble protein of this fraction.Correspondingly, northern analysis showed overexpression of phyA mRNA inthese phytase gene transformants and no signal was viewed in the controlcells (See FIG. 3).

In order to optimize phytase protein expression, the time course and theeffects of a series of factors on the expression were studied. Thesefactors included incubation temperature (30 and 37° C.), medium pH (4.0,5.0, 6.0, 7.0, 8.0, and 9.0), anaerobiosis (adding sterile mineral oilon the top of the growing cells), inorganic phosphate level in themedium (Dassa, E. et al., “The Acid Phosphatases with Optimum pH of 2.5of Escherichia coli,” J. Bio. Chem., 257:6669-76 (1982), which is herebyincorporated by reference), and sodium phytate (0, 0.1, 0.2, 0.3, 0.4,and 0.5 mM). Results indicated that expression of phytase protein wasaccumulated linearly with time for the first six hours after induction(See FIG. 4). Thereafter, the expression remained relatively unchangedalthough bacterial cells continued to grow. Only medium pH and sodiumphytate concentration significantly affected the phytase proteinexpression. Maximum protein was shown at pH 6.0 and 0.3 mM of sodiumphytate, in which phytase protein was increased from 3.8 to 9.6% of thetotal soluble protein.

No phytase activity was detected extracellularly or intracellularly.This may not be completely unexpected, because the native phytase fromA. niger is a glycoprotein with a size of 70-80 kDa (Hartingsveldt etal., “Cloning, Characterization and Overexpression of thePhytase-Encoding Gene (phyA) of Aspergillus Niger,” Gene 127:87-94(1993), which is hereby incorporated by reference). The proteinexpressed in the E. coli system of this study had a size ofapproximately 55 kDa. Presumably, the lack of glycosylation of theprotein and other necessary post-translational modifications duringsecretion would preclude phytase activity.

Example 3 Expression of PhyA in S. lividans

Heterologous genes have been expressed in S. lividans, and the resultingproducts secreted into the medium with enzymatic activity (Ghangas, G.S. et al., “Cloning of the Thermomonospora Fusca Endoglucanase E2 Genein Streptomyces Lividans: Affinity Purification and Functional Domainsof the Cloned Gene Product,” Appl. Environ. Microbiol., 54:2521-26(1988); Wilson, D. B., “Biochemistry and Genetics of ActinomyceteCellulases,” Crit. Rev. Biotechnol., 12:45-63 (1992); Jung, E. D. etal., “DNA Sequences and Expression in Streptomyces Lividans of anExoglucanase Gene and an Endoglucanase Gene from Thermomonospora Fusca,”Environ. Microbiol., 59:3032-43 (1993), which are hereby incorporated byreference). Similarly, phyA gene was expressed in S. lividans and theprotein was introduced into the medium, as shown in a specific band inthe SDS-PAGE analyzed medium samples (See FIGS. 5 and 6). This suggestedthat the signal peptide from endoglucanase E2 gene of T. fusca was ableto lead phytase protein out of the cell. This protein was 57 kDa andrepresented 16.2% of the total protein in the medium. Changing medium pHto 6.0 and adding 0.3 mM of sodium phytate in the medium improved theprotein yield to 20.3% of the total protein. Because phytase protein wassecreted into the medium in such a high level, it should be easy topurify and used effectively for a variety of purposes such as producingphytase antibody. Once again, no increased phytase activity was foundeither in the medium or in the lysed cells. Although the protein sizeincreased a little bit (2-3%) compared to the one expressed in E. coli,presumably due to glycosylation of phytase protein in this expressionsystem, there was still no phytase activity.

Example 4 Expression of PhyA in S. cerevisiae

Three different signal peptides were used to compare the efficiency inleading the expressed protein out of the cells (See Table 2). Thephytase activity was substantially increased in the Sabouraud-raffinosemedium growing the transformants of pYEP1 and pYPP1, but not pYXP1.Visible phytase protein was shown by SDS-PAGE 20 hours after induction(FIG. 7).

The expression of transformants of pYEP1 and pYPP1 were determined inthree different types of medium: Sabouraud-raffinose (Li, X. L. et al.,“Expression of Aureobasidium Pullulans XynA in, and Secretion of theXylanase From, Saccharomyces Cerevisiaea,” Appl. Environ. Microbiol.,62:209-13 (1996), which is hereby incorporated by reference),Sabouraud-glycerol, and a modified general-purposed YEPD medium. As totransformants of pYEP1, similar phytase activity was expressed in theSabouraud-raffinose and Sabouraud-medium, but there was no activitydetected in the YEPD medium. In contrast, phytase activity in the mediumcultured with transformants of pYPP1 varied greatly with the differenttypes of medium. The activity was enhanced to 375 mU/ml whenSabouraud-glycerol medium was used. The activity was further increasedto 1675 mU/ml, when the medium was changed to YEPD (See Table 3). Whilethe YEPD medium was much cheaper than the Sabouraud-raffinose medium,the phytase yield was increased more than ten-folds. Thus, the putativesignal peptide from the fungal phytase gene achieved the most efficientexpression of the extracellular phytase activity. Nearly all the proteinproduced was secreted into the YEPD medium, because very little activitywas detected in the yeast cells. The time course of the phytaseexpression in this system was shown in FIG. 8.

TABLE 3 Phytase activity expressed from transformant with pYPP1 indifferent media Hours after induction (mPU/ml)¹ Medium 0 10 15Sabouraud-raffinose 22 136 146 Sabouraud-glycerol 6 174 375 YEPD 18 12381675 ¹The phytase activity was detected in the supernatant of cellculture of the three media 0, 10, and 15 hours after induced by addinggalactose. See text for definition of phytase units.

A variety of microorganisms including bacilli, yeasts, and filamentousfungi have phytase activity, while A. niger NRRL3135 strain produces thehighest activity (340 mU/ml, Shieh, T. R. et al., “Survey ofMicroorganisms for the Production of Extracellular Phytase,” Appl.Environ. Microbiol., 16:1348-51 (1968), which is hereby incorporated byreference). Schwanniomyces castellii CBS 2863 has the highest phytaseactivity among 21 yeast strains (140 mU/ml, Lambrechts, C. et al.,“Utilization of Phytate by Some Yeasts,” Biotechnology Letters, 14:61-6(1992), which is hereby incorporated by reference). Clearly, therecombinant yeast strain transformed with pYPP1 in the present studyproduced much higher phytase activity (1675 mU/ml) than A. niger(4-fold) and S. castellii CBS 2863 (11-fold). Maximum phytase productioncan be obtained in the system by optimizing the incubation conditionsand modifying the plasmid cassettes (Demolder, J. W. et al., “EfficientSynthesis of Secreted Murine Interleukin-2 by Saccharomyces Cerevisiae:Influence of 3′-Untranslated Regions and Codon Usage,” Gene, 111:207-13(1992), which is hereby incorporated by reference).

The high level of phytase activity expression in S. cerevisiae was mostlikely due to the sufficient glycosylation of phytase protein and otherpost-translational modifications by yeast. After the medium supernatantwas concentrated and subjected to SDS-PAGE analysis, there was a bandwith approximately 110 kDa (See FIGS. 7 and 9), which was larger thanthe size of the native protein from A. niger (Hartingsveldt, W. van. etal., “Cloning, Characterization and Overexpression of thePhytase-Encoding Gene (phyA) of Aspergillus Niger,” Gene 127:87-94(1993), which is hereby incorporated by reference). Northern analysisconfirmed the specific overexpression of phyA mRNA (See FIG. 10). Theseresults indicated that the yeast system was efficient to overexpressactively extracellular phytase enzyme. Yeast system has severaladvantages over bacteria or other systems such as A. niger(Hartingsveldt, W. van. et al., “Cloning, Characterization andOverexpression of the Phytase-Encoding Gene (phyA) of AspergillusNiger,” Gene 127:87-94 (1993), which is hereby incorporated byreference). It carries out post-translational modifications, includingproper folding, glycosylation, disulfide bond formation, andproteolysis, during the translocation of proteins through theendoplasmic reticulum and the cell membrane. The secretion of proteinsis facilitated by hydrophobic short signal peptides at the N-terminalregions of the protein precursors (Li, X. L. et al., “Expression ofAureobasidium Pullulans XynA in, and Secretion of the Xylanase From,Saccharomyces Cerevisiaea,” Appl. Environ. Microbiol., 62:209-13 (1996),which is hereby incorporated by reference). Proteins secreted by yeastcells are protected from aggregation and protease degradation. Mostimportantly, enzyme proteins produced by S. cerevisiae are easilypurified, because it secretes only a few proteins. Considering thewell-known safety of yeast products to both human beings and animals,this system is of great potential for human food and animal feedindustry.

Example 5 Properties of the PhyA Phytase Overexpressed in Saccharomycescerevisiae

The overexpressed phytase from transformants of pYPP1 plasmid wasconcentrated and used to study its property (See Table 4). The enzymeshowed two optimum pH ranges: 2 to 2.5 and 5.0 to 5.5. However, enzymeactivity at pH 2 to 2.5 was only 60% of the activity at pH 5 to 5.5.There was no activity detected at either pH 1 or 8. The optimum pH wasvirtually the same as the phytase from A. niger (Simons et al.,“Improvement of Phosphorus Availability by Microbial Phytase in Broilersand Pigs,” Br. J. Nutr., 64:525 (1990), which is hereby incorporated byreference), thus active function in hydrolysis of phytate-P in thegastrointestinal tracts would certainly be expected. The optimumtemperature of the enzyme was 60° C., while the current one on themarket produced by Gist-Brocades is 55° C. (BASF, 1996). More than 80%of the activity remained at 50 to 55° C., but little activity wasdetected at 75 or 80° C. Heating the enzyme for 15 min at 37 and 80° C.,the remaining activity for the expressed yeast phytase of the presentinvention was 100 and 63%, respectively, and for BASF Gist-Brocadesphytase was 100 and 52%, respectively. The differences between the twoenzyme sources at any given temperature were significant (See Table 5).Thus, the yeast phytase appeared to be more heat stable than the currentcommercial phytase product.

TABLE 4 Characteristics of the overexpressed phytase in yeast¹ OptimumpH² pH 1.0 2.0 2.5 3.0 4.0 5.0 5.5 6.0 8.0 Relative .5^(e) ± .2 59.7^(c)± 3 64.8^(c) ± 6 48.1^(d) ± 4 81.0^(b) ± 5 100.0^(a) ± 1 95.0^(a) ± 666.3^(c) ± 1 .8^(e) ± .4 Activity (%) Optimum Temperature³ ° C. 25 37 4550 55 60 75 80 Relative 24.2^(e) ± .8 44.6^(d) ± 3 63.9^(c) ± 8 83.6^(b)± 2 89.8^(b) ± 4 100.0^(a) ± 4 .6^(f) ± .1 .9^(f) ± .2 activity (%)¹Data are means of relative activity ± standard deviation (n = 4). Meansin a row with different superscript letters differ (P < 0.05). Thegeneral linear model of the statistical analysis system (1988) was usedto analyze the main treatment effects as randomized complete designs andBonferroni t-test was used for multiple treatment mean comparison.Significance level was set as P < 0.05. ²The activity was assayed at 37°C. (see context for phytase unit definition). Different buffers wereused: 0.2 mM glycine-HCI buffer for pH 1.0 to 3.5; 0.2 mM sodium citratebuffer for pH 4.0 to 6.5; and 0.2 mM Tris-HCI buffer for pH over 7.³Optimum temperature was determined at pH 5.5 (0.2 mM sodium citratebuffer).

TABLE 5 Comparison of the thermostability of overexpressed phytase inyeast and Gist-Brocades phytase produced by A. niger ^(1,2) Relativeactivity, % 37° C. 80° C. Yeast phytase 100^(a) ± 1 63^(b) ± 1 A. nigerphytase 100^(x) ± 3 52^(y) ± 2 P³ < .03 ¹Data are means of relativeactivity ± standard deviation (n = 3). Means in a row with the differentsuperscript letters differ (P < 0.05). The general linear model of thestatistical analysis system (1988) was used to analyze the maintreatment effects as randomized complete designs and Bonferroni t-testwas used for multiple treatment mean comparison. Significance level wasset as P < 0.05. ²The enzyme was heated for 15 minutes at differenttemperatures before reacting at 37° C. and pH 5.5. ³Significance (Pvalues) of t-test between the activity of the two phytases at eachtemperature setting.

Although it is unclear how such improvement in thermostability isrelated to different post-translational modifications (folding,cleavage, glycosylation, etc.), (Li, X. L. et al., “Expression ofAureobasidium Pullulans XynA in, and Secretion of the Xylanase From,Saccharomyces Cerevisiaea,” Appl. Environ. Microbiol., 62:209-13 (1996),which is hereby incorporated by reference), it is certainly advantageousto have more thermostable phytase enzyme that can hopefully be resistantto the heat during feed pelleting, which is a problem with the currentGist-Brocades phytase.

Example 6 In Vitro Hydrolysis of Phytate-P from Corn, Soy, and WheatMiddlings by the Expressed Yeast Phytase

The expressed yeast phytase released phytate-P from corn and soybeanmeal as effectively as the Gist-Brocades phytase based on per unitactivity (See Table 6). As expected, the hydrolysis of phytate-P was afunction of time and activity dosage. The expressed yeast phytase wasalso effective in releasing phytate-P from wheat middling, indicatingits great potential in bread fermentation. Because the wheat middlingused in this study contained much higher intrinsic phytase activity thancommonly used wheat flour, much greater effect of the expressed yeastphytase on improving flour phytate-P hydrolysis and in trace elementreleasing would be expected, when it is used in a bakery (Hall, M. N. etal., “The Early Days of Yeast Genetics,” Cold Spring Harbor LaboratoryPress (1993), which is hereby incorporated by reference).

TABLE 6 Free phosphorus released from corn, soybean meal (SBM), andwheat middlings by overexpressed yeast phytase and fungus A. nigerphytase in vitro¹ 250 Yeast phytase (fungus (PU/kg) 0 100 250 500 1000phytase) Free phosphorus (mg/g) Corn: 1 hour  .23^(d) ± .03   .64^(c) ±.08 1.14^(b) ± .18 1.46^(a) ± .04 1.54^(a) ± .04 1.16^(b) ± .15 4 hour .36^(c) ± .02  1.26^(b) ± .04 1.60^(a) ± .03 1.66^(a) ± .06 1.72^(a) ±.04 1.68^(a) ± .04 SBM: 1 hour  .68^(d) ± .01 1.18^(cd) ± .02 1.62^(c) ±.18 2.48^(b) ± .32 3.13^(a) ± .19 1.68^(c) ± .2 4 hour .73^(d)  1.67^(c)2.69^(b) 3.41^(a) 3.71^(a) 2.78^(b) Wheat Middlings: 1 hour 3.56 ± .39 4.11 ± .64 4.67² ± .05 4 hour 5.63 ± .5  6.02 ± .48 6.38² ± .07 ¹Eachsample of 5 g was stirred in 20 ml of 0.2 mM sodium citrate buffer at37° C. for 1 or 4 hours. The supernatant was obtained by spinning for 15minutes at 8000 g. After going through Whatman 541 filter paper, thesample was subjected to free P assay by the method of Chen, P. S. etal., “Microdetermination of P,” Anal. Chem., 28: 1756-58 (1956), whichis hereby incorporated by reference. Data in the table are means ofrelative activity ± standard deviation(n = 4). The General Linear Modelof the Statistical Analysis System (1988) was used to analyze the maintreatment effects as randomized complete designs and Bonferroni t-testwas used for multiple treatment mean comparison. Significance level wasset as P < 0.05. A significant difference existed between 1 and 4 hourfor every feed at each dose of enzyme as analyzed by t-test. Means in arow with different superscript letters differ (P < 0.05). ²n = 2

The overexpression of Aspergillus niger phytase (phyA) in Escherichiacoli, Streptomyces lividans, and Saccharomyces cerevisiae were comparedto develop an efficient and simple system to produce phytaseeconomically. A 55 kDa soluble intracellular protein, representing 9.6%of the total soluble protein, was expressed in E. coli by usingpET25b(+) system. A 57 kDa extracellular protein, representing 20.3% ofthe total protein in the medium, was expressed in S. lividans by using ashuttle plasmid containing the pLT1 promoter and Spell leading peptideof endoglucanase E2. No increase in phytase activity was shown in eitherexpression system, presumably due to the lack of glycosylation and othernecessary post-translational modification. In contrast, highextracellular phytase activity was produced in S. cerevisiae transformedwith phyA gene. Three different signal peptides and three differenttypes of medium were compared to identify the best expression vector andcondition. Use of the signal peptide Sphy from phyA gene and YEPD mediumproduced the highest extracellular phytase activity. The overexpressedphytase in yeast was approximately 110 kDa, had two pH optima: 2.0 to2.5 and 5.5 to 6.0, and the optimum temperature was at 60° C.

Example 7 Methods and Materials for Expression of phyA in Pichia

Host and vector. An EasySelect™ Pichia Expression Kit was purchased fromInvitrogen (San Diego, Calif.). The kit provides hosts and vectors toexpress the gene either intracellularly or extracellularly, in strainsof either Mut⁺ or Mut^(s) (Methanol utilization normal or slow). X33 wasused as a Mut⁺ strain and KM71 as a Mut^(s) strain. Two vectors wereused, pPICZ B (3.3 kb) and pPICZαA (3.6 kb), both use AOX1 as thepromoter.

Construction of the Expressing Vectors. To compare the effect ofdifferent signal peptides on the expression of PhyA in Pichia system,two constructs were prepared. First, a 1.4 kb EcoRI-KpnI fragment,containing the PhyA sequence encoding the mature phytase protein, wasligated into pPICZαA. In this plasmid (pPICZα-phyA), PhyA was led by analpha-factor, a very general-used signal peptide from Saccharomycescerevisiae. Second, a 1.4 kb KpnI-XbaI fragment of pYPP1 was ligatedinto the vector (the coding region of phyA was led by its own signalpeptide that was very effective in secreting the expressed phytase inSaccharomyces cerevisiae.)

Transformation and expression. The confirmed constructs were linearizedby PmeI and transformed into GS115 and KM71, by EasyComp™ provided bythe kit. Neocin™ was used to select the positive colonies. After asingle colony was inoculated into 10 ml of MGY medium and grown to OD₆₀₀of 2-6 at 30° C., the cells were collected by centrifugation andresuspended into 10 ml of MMY medium (containing 0.5% of methanol). Thesamples were collected every 12 or 24 h after induction. The cells wereseparated from the supernatant and lysed with glass beads in breakingbuffer. Phytase activity in the supernatant and cells was assayed asdescribed previously. SDS-PAGE and Western blot were conducted todetermine the size and relative amount of the expressed protein.

Example 8 PhyA Phytase Activity in Pichia

The expression construct using alpha-factor as the signal peptide forphyA was transformed into two Pichia strains. KM71 is a methanolutilization slow strain, while X33 is a Pichia wild-type utilizingmethanol efficiently. The screening and incubation were conducted in 10ml shake flasks under 29-30° C. For the transformants of KM71, 19 out of20 picked colonies had extracellular phytase activity greater than 6units/ml of culture supernatant after induction for 24 hours. Colony No.13 showed the highest activity of 26 units/ml after incubated for 108hours. For the transformants of X33, all colonies (20/20) had more than10 units/ml after induced for 24 hours. One of the colonies (#101)produced phytase activity of 65 units/ml of supernatant. A time coursestudy of the phytase expression in KM71 and X33 was summarized in FIG.11. Despite the difference of these two strains in utilizing methanoland, therefore, the ability in expressing phytase, it was found thatalpha-factor was correctly processed by yeast cells. Besides, almost allof the expressed protein was secreted into the medium since not morethan 5% of the total activity expressed was found intracellularly.

Effects of inorganic phosphorus and pH of media on the phytaseexpression were studied in the media (BMGY and BMMY) using a phyArecombinant of X33 (#101). The medium containing 50 mM phosphateproduced the highest phytase activity, 66 units/ml at 168 hours afterinduction. By including 50 mM phosphate in the media, the effect ofdifferent pH of this buffer (3, 4, 5, 6, 7, and 8) on expression wasalso studied. When the pH was 6, this X33 transformant produced 75 unitsphytase/ml supernatant. Based on the protein concentration and SDS-PAGEanalysis, the expression phytase protein yield was estimated to bebetween 3 to 4 mg/ml.

Example 9 Properties of the PhyA Phytase Expressed in Pichia

Molecular size and deglycosylation of the expressed phytase. After thesupernatant of the medium inoculated with the phyA transformant wassubjected to SDS-PAGE, a strong band around 95 kDa was seen (FIG. 12).This was almost the only viewed protein in the supernatant. Theexpressed phytase reacted efficiently with the rabbit polyclonalantibody raised against purified native A. niger phytase. This indicatedthat the immunoreactivity of the expressed phytase was essentially thesame as that of the native phytase from A. niger. The size was decreasedto 50 kDa by deglycosylation using Endo H. The phyA antibody alsoreacted with the deglycosylated phytase. In addition, deglycosylation,conducted under native conditions, reduced the phytase activity about15%, indicating that glycosylation was important for the activity of thephytases. Moreover, glycosylation affected the thermostability of theenzymes (FIG. 13).

Northern analysis. As showed in FIG. 14, a 1.3 kb phyA DNA probehybridized with the mRNA of the induced transformants from both KM71(#13) and X33 (#101). Response was also seen from the transformantsprior to induction. Probably, the expression of phyA in this system wasnot controlled strictly at the level of transcription.

Optimal pH and temperature and phytate-phosphorus hydrolysis. Similar toA. niger phytase, the expressed phytase had two optimum pH, 2.5 and 5.5(FIG. 15). The optimum temperature of the expressed phytase was 60° C.(FIG. 16). When the expressed phytase was incubated with soy samples at100, 200, 400, 800 mU/g of sample at 37° C., phosphorus was released ina linear fashion with the phytase dose (FIG. 17).

Example 10 Methods and Materials for Overexpression of E. coli appA Genein Saccharomyces cerevisiae

Gene and Protein. This gene, originally defined as E. coli periplasmicphosphoanhydride phosphohydrolase (appA) gene, contains 1,298nucleotides (GeneBank accession number: M58708). The gene was firstfound to code for an acid phosphatase protein of optimal pH of 2.5(EcAP) in E. coli. The acid phosphatase is a monomer with a molecularmass of 44,644 daltons. Mature EcAP contains 410 amino acids (Dassa, J.et al., “The Complete Nucleotide Sequence of the Escherichia coli GeneappA Reveals Significant Homology Between pH 2.5 Acid Phosphatase andGlucose-1-Phosphatase,” J. Bacteriology, 172:5497-5500 (1990), which ishereby incorporated by reference). Ostanin, K. et al. (“Overexpression,Site-Directed Mutagenesis, and Mechanism of Escherichia coli AcidPhosphatase,” J. Biol. Chem., 267:22830-36 (1992), which is herebyincorporated by reference), overexpressed appA in E. coli BL21 using apT7 vector and increased its acid phosphatase activity by approximately400-fold (440 mU/mg protein).

The gene and a host E. coli strain CU 1869 (No. 47092) were purchasedfrom ATCC. The gene, an insert of 1.3 kb, was transformed into E. colistrain BL21 (no. 87441) using an expression vector pAPPA1 (Ostanin, K.et al., “Overexpression, Site-Directed Mutagenesis, and Mechanism ofEscherichia coli Acid Phosphatase,” J. Biol. Chem., 267:22830-36 (1992),which is hereby incorporated by reference).

Host and Vector. The vector for overexpressing appA gene inSaccharomyces cerevisiae was pYES2 and the host was INVScI (Invitrogen,San Diego, Calif.).

Construction of the Expression Vector. Initially, a 1.3 kb XbaI fragmentwas isolated from pAPPA1. This fragment contained the appA gene with itsown signal peptide. After being ligated into the XbaI site of pYES2, theconstruct (PYES2-appA) was transformed into Saccharomyces cerevisiae.But, no phytase activity was increased in either extra- orintra-cellular parts compared to the controls. pAPPA1 and pYPP1 (PhyAand its signal peptide in pYES2) were cotransformed into the yeaststrain. Again, no increase in phytase activity due to pAPPA1 wasdetected in the media or the yeast cells.

Two primers were synthesized to construct the signal peptide of PhyAgene with the coding region of appA gene. One was 80 bp long containingthe PhyA signal peptide and a KpnI site at 5′ end: GGG GTA CCA TGG GCGTCT CTG CTG TTC TAC TTC CTT TGT ATC TCC TGT CTG GAG TCA CCT CCG GAC AGAGTG AGC CGG AG (SEQ ID No. 9). The other primer was 24 bp long, with anEcoRI site at its 3′ end: GGG AAT TCA TTA CAA ACT GCA GGC (SEQ ID No.10). The PCR was run for 25 cycles, with 1 min denaturing at 95° C., 1min annealing at 58° C., and 1 min chain extending at 72° C. A 1.3 kbfragment was amplified, digested, and ligated into pYES2. After theinsert was confirmed by restriction mapping, the construct(pYES2-SphyA-appA) was transformed into INVScI by lithium acetatemethod.

Expression. The selected transformants were inoculated into YEPD medium.The expression was induced by adding galactose into the culture afterOD₆₀₀ reached 2, as described previously. The cells were harvested 15 or20 h after induction.

Activity Assay. Acid phosphatase activity was assayed at 37° C. in 25 mMglycine-HCI buffer (pH 2.5), using p-nitrophenyl phosphate as thesubstrate (stock 250 mM). Reaction buffer of 1.7 ml was added into 0.1ml samples. After they were incubated for 5 min in a 37° C. waterbath,0.2 ml of prewarmed substrate was added and mixed. The reaction solutionwas transferred into a prewarmed cuvette and incubated for 2 min in a37° C. spectrophotometric compartment. The released p-nitrophenol wasread continuously for 5 min at 405 nm for enzyme activity calculation.

In vitro study. Soybean meal (5.0 g) was suspended into 20 ml of 20 mMcitrate buffer, pH 5.5, mixed with 200 mU of phytase, incubated at 37°C. for 4 h with continuous shaking. After chilling on ice for 10 min,the slurry was transferred into a centrifuge tube and spun for 15 min at15,000×g. The supernatant was used to determine free phosphorus.

Example 11 Quantitation of Phytase Activity from Overexpression of E.coli appA Gene in Saccharomyces cerevisiae

The intracellular acid phosphatase activity in the appA overexpressed E.coli (pAPPA1) was 440 mU/mg protein. Unprecedently, an intracellularphytase activity greater than 4900 mU/mg protein was found in thetransformed strain. But, there was only minimal phytase activity in thecontrol (BL21). Thus, this acid phosphatase gene also codes for aphytase. The appA gene sequence was aligned with that of PhyA and foundthat these two genes shared 23% of identity.

Transforming INVScI with the construct of pYES2-Sphy-appA (led by thesignal peptide of PhyA) produced extracellular phytase activity in thesupernatant that was 2,000-fold greater than those of the wild type orof the transformant containing appA gene plus its own signal peptide(See Table 7).

TABLE 7 Extracellular phytase activity in transformants of appA genewith different signal peptides Activity (mU/mg Construct Signal Activity(mU/ml) protein) PYES-appA appA Undetectable Undetectable pYES2-SphyA-PhyA 1,158 445 appA

The effects of medium (YEPD) inorganic phosphorus, phytate, pH, andtemperature on the expression of phytase activity by pYES2-Sphy-A-appAare presented in Table 8. The highest phytase activity was 2,286 mU/ml(633 mU/mg protein) at the optimal condition.

TABLE 8 Effect of different conditions in the YEPD medium on phytaseactivity expression of pYES2-SphyA-appA in yeast. Medium ConditionsActivity (mU/ml) Phosphorus, mg/100 ml 0 1402 1 714 5 722 10 456 Sodiumphytate, g/100 ml 0 870 0.1 1019 1.0 1748 pH 5.0 892 7.0 996 8.0 2286Temperature, ° C. 25 312 30 1036 37 996The thermostability of the overexpressed extracellular phytase activityproduced by the yeast transformant was greater than that of theintracellular phytase produced by E. coli transformed with pAPPA1 (SeeTable 9). Heating the extracellular phytase for 15 min at 80° C.resulted in 30% of loss of its phytase activity, while almost all thephytase activity from E. coli was lost under the same condition.

TABLE 9 Effect of heating different sources of phytases under 80° C. for15 min on their activities Phytase Relative activity after heating, %appA in E. coli 0.1 appA in S. cerevisiae 69 PhyA in S. cerevisiae 66BASF phytase 50

Comparisons of the effect on releasing phosphorus from soybean meal byphytases (200 mU) of E. coli, overexpressed AppA in yeast, and BASF arepresented in Table 10. The results indicate that all three sources ofphytases released phytate-phosphorus effectively from soybean meal.

TABLE 10 Free phosphorus released from soybean meal by different sourcesof phytases Phytase Phosphorus (mg/g) appA in E. coli 1.11 appA in S.cerevisiae 0.69 BASF 0.87

E. coli appA (acid phosphatase) gene when expressed in Saccharomycescerevisiae produces extracellular phytase activity in the media that wasmore than 2,000-fold greater than the control. The overexpressed phytaseeffectively releases phytate-phosphorus from soybean meal, and seems tobe more thermostable than the presently available commercial phytase orthe intracellular phytase produced in E. coli by the same gene (appA).

Example 12 Methods and Materials for Overexpressing the E. coli appAGene Encoding an Acid Phosphatase/Phytase in Pichia pastoris

Gene and Protein. The appA gene and the host E. coli strain CU1867 (No.47092) were obtained from ATCC. The gene, an insert of 1.3 kb, wastransformed into E. coli strain BL21 (No. 87441) using an expressionvector pAPPA1 (Ostanin, K. et al., “Overexpression, Site-DirectedMutagenesis, and Mechanism of Escherichia coli Acid Phosphatase,” J.Biol. Chem., 267:22830-36 (1992), which is hereby incorporated byreference).

Host and Vector. An EasySelect™ Pichia Expression Kit was obtained fromInvitrogen (San Diego, Calif.). The kit provides hosts and vectors toexpress the gene either intracellularly or extracellularly in awild-type strain (X-33). Two vectors were used, pPICZ B (3.3 kb) andpPICZαA (3.6 kb), both use AOX1 as the promoter.

Construction of the Expression Vector. Two primers were used to amplifythe appA gene from pAPPA1 and two restriction sites EcoRI and KpnI wereproduced at the 5′ and 3′ ends, respectively.

Upstream primer: GGA ATT CCA GAG TGA GCC GGA (SEQ ID No. 11)Downstream primer: GGG GTA CCT TAC AAA CTG CAC G (SEQ ID No. 12)Template: pAPPA1 DNA isolated from ATCC 87441

PCR was run for 30 cycles, with 1 min denaturing at 94° C., 1 minannealing at 55° C., and 1 min chain extending at 72° C. A 1,245base-pair fragment was amplified, digested with EcoRI and KpnI, andligated (16° C. overnight) into pPICZ B (3.3 kb) and pPICZαA (3.6 kb).The ligation was confirmed by restriction mapping after transforming theconstructs into DH5α.

Transformation of the construct into Pichia (X33). For eachtransformation, 100 μg of plasmid DNA was prepared and linearized bydigesting with PmeI. After linearization, the DNA was purified andresuspended into 10 μL of sterile, deionized water. Half amount of theDNA was actually used for each transformation. Electroporation and theEasyComp chemical kit (Invitrogen) were both used to transform the DNAinto X33. In the case of electroporation, an Electro Cell Manipulator(ECM 600, Gentromics, BTX Instrument Division, San Diego, Calif. 92121)and 2 mm cuvettes were used. The resistance was 186 Ohm, the chargingvoltage was 1.5 kilovolts, and the actual charging length wasapproximately 7 milliseconds. The electroporated cells were incubated onYPD agar plates containing 100 mg Zeocin/mL at 30° C. for 2-4 days forcolony growth. In the case of chemical transformation, cells were grownon YPDS agar plates containing 100 mg Zeocin/mL. Compared with theelectroporation, the chemical method had lower transformationefficiency.

Expression. Single colonies were inoculated into 10 ml of MGY medium (30ml tube) and grown (16-18 h) to OD₆₀₀ of 5-6 at 28-30° C. in a shakingincubator (200 rpm). The cells were collected by centrifugation (2,000rpm) and resuspended into 10 ml of BMMY medium (containing 0.5% ofmethanol) to induce the expression. The samples (200 μL) were collectedevery 12 or 24 h after induction. Methanol (100%) was added at 100 μLevery 24 to maintain a concentration of 0.5-1% in the media.

Assays. The cells were separated from the media (supernatant) and lysedwith glass beads in breaking buffer. Extracellular phytase activity inthe supernatant and intracellular phytase activity in the lysed cellswere assayed as described previously (0.2 M citrate buffer, pH 5.5 under37° C. using 10 mM sodium phytate). Acid phosphatase activity wasassayed at 37° C. in 25 mM glycine-HCI buffer (pH 2.5), usingp-nitrophenyl phosphate as the substrate (stock 250 mM). Reaction bufferof 1.7 ml was added into 0.1 ml samples. The released p-nitrophenol wasread continuously for 5 min at 405 nm for enzyme activity calculation.SDS-PAGE (12%) was conducted to determine the size and relative amountof the expressed protein. The optimal pH and temperature of theexpressed phytase were determined as described in the results.

In vitro study. Soybean meal (5.0 g) was suspended into 20 ml of 20 mMcitrate buffer, pH 5.5, mixed with different levels of phytase, andincubated at 37° C. for 4 h with continuous shaking. After being chilledon ice for 10 min, the slurry was transferred into a centrifuge tube andspun for 15 min at 15,000×g. The supernatant was used to determine freephosphorus.

Example 13 Colony Phytase Activity Screening for Phicia pastorisOverexpressing the E. coli appA Gene

Wild-type Pichia X33 produces minimal phytase activity intracellularly(<0.03 U/mg protein) or extracellularly (<0.05 U/mL). The X33 cellstransformed with the appA gene inserted into pPICZB (without theα-factor and presumably produces intracellular phytase) did not show anyincrease in phytase activity (extracellular, 0.2 U/mL and intracellular,0.05 U/mg protein).

Transforming X33 cells with the construct of pPIZαA-appA (led by thesignal peptide of α-factor) produced extracellular phytase activity inthe media. Initially, 72 colonies were screened. Only two colonies hadactivity <1 U/mL 40 hours after induction. Most of the colonies hadactivity ranging from 10 to 20 U/mL 40 hours after induction. All of the70 colonies had phytase activity >80 U/mL 118 hours after induction. Thehighest phytase activity so far detected was 215 U/mL, 192 hours afterthe induction (See Table 11).

TABLE 11 Range of extracellular phytase activity in X33 coloniestransformed with pPIZαA-appA 40 and 118 hours after induction. 118 hoursafter Number of Colonies 40 hours after induction induction 2 <1 U/mL 61 to 10 U/mL 36 11 to 20 U/mL 28 >20 U/mL 70 >80 U/mL

Phytase and acid phosphatase activities in the transformant expressing215 U phytase activity/mL were compared with those of the wild-type ofX33 (192 hours after induction) (See Table 12). Almost all of theexpressed phytase protein was secreted from the cells, indicating thatα-factor was a very effective signal peptide for phytase secretion.

TABLE 12 Phytase and acid phosphatase activities in the pPIZαA-appAtransformant and the wild-type of X33 192 hours after induction.Wild-type X33 pPIZαA-appA transformant Extracellular IntracellularExtracellular Intracellular U/mL U/mg protein U/mL U/mg protein Phytase0.05 0.03 215 0.5 Acid 0.01 0.002 5.88 0.9 phosphatase

Transformants of E. coli with the same acid phosphatase appA gene hadintracellular phytase activity of 5 U/mg protein (Ostanin et al.,“Overexpression, Site-Directed Mutagenesis, and Mechanism of Escherichiacoli Acid Phosphatase,” J. Biol. Chem., 267:22830-36 (1992), which ishereby incorporated by reference). Transforming PhyA gene in A. nigerproduced an extracellular activity of 7.6 U/ml (Hartingsveldt et al.,“Cloning, Characterization and Overexpression of the Phytase-EncodingGene (phyA) of Aspergillus Niger,” Gene 127:87-94 (1993), which ishereby incorporated by reference). Compared with these results, thephytase expression system in Pichia is a very efficient expressionsystem.

Example 14 Time-Course of Phytase Expression

There was a linear increase in extracellular phytase activity in themedia almost in all of the selected colonies up to 192 hours afterinduction. FIG. 18 summarized the activity changes of five selectedcolonies from 24 to 163 hours after induction.

Example 15 Effects of Medium pH on the Expression of Phytase (Colony#23, Activity 136 U/mL at 186 h)

Using 0.1 M phosphate buffered media, the effects of different pH on theproduction of extracellular phytase in the transformants were studiedagainst a control medium without buffer (pH 7.0). The medium buffered topH 6 produced the highest phytase activity (See FIG. 19).

Example 16 Size of the Expressed Extracellular Phytase

Using SDS-PAGE (12% gel) analysis, a clear band was noticed in themedium supernatant of culture inoculated with three different colonies(See FIG. 20). The size was around 55 kDa, probably partiallyglycosylated. Because the expressed protein represented almost the onlyvisible band in the supernatant, it would be convenient to collect theenzyme product without the need for a tedious purification.

Example 17 Optimum pH and Temperature of the Expressed ExtracellularPhytase (Colony #23)

The optimum pH of the expressed phytase was 2.5 to 3.5 (See FIG. 21).This is significantly different from that of phyA phytase either from A.niger (BASF) or our other expression systems. It is ideal for phytasefunction at the stomach pH.

The optimum temperature of the expressed enzyme was 60° C. (See FIG.22).

Example 18 Effect of the Expressed Phytase on Phytate-PhosphorusHydrolysis from Soybean Meal

This overexpressed E. coli phytase (Colony #23) effectively hydrolyzedphytate-phosphorus from soybean meal (See FIG. 23). The release of freephosphorus in the mixture was linear from 0 to 800 mU of phytase/g offeed.

Example 19 Effects of the Expressed E. coli AppA Phytase by Pichiapastoris on Phytate Phosphorus Bioavailability to Weanling Pigs

To determine the nutritional values of the expressed E. coli phytase byPichia in swine diets, the efficacy of this new phytase was comparedwith those of inorganic phosphorus or the commercially availablemicrobial phytase (Natuphos™, BASF Corp., Mt. Olive, N.J.). Forty-eightweanling pigs were selected from multiparous sows at Cornell SwineResearch Farm. The pigs were weaned at 21 days of age and fed acommercial creep feed until day 28. They were then placed two per penwith six pens assigned randomly per treatment. The pigs were given twoweeks to adjust to the corn-soybean meal basal diet (Table 13).

TABLE 13 Formulation of the Experiment Diets for Pigs. +C % −C % YP % MP% Ingredient diet diet diet diet Corn 60.5 61.57 61.07 61.07 WheyProtein Concentrate 3 3 3 3 SBM 44% 30 30 30 30 Corn Oil 3 3 3 3 Lime0.8 0.93 0.93 0.93 Di-calcium phosphate 1.2 0 0 0 Vitamin and Mineralpremix 0.5 0.5 0.5 0.5 ECAP premix 0 0 0.5 0 MP premix 0 0 0 0.5 Salt0.5 0.5 0.5 0.5 CSP 250 0.5 0.5 0.5 0.5 Total 100 100 100 100 CP 20.620.6 20.6 20.6 Ca 0.73 0.47 0.47 0.47 P_(total) 0.6 0.39 0.39 0.39 Note:All premixes use corn as the carrier Vitamin and Mineral Premixsupplies: 2,540 IU Vit. A, 660 IU Vit. D, 15 IU Vit. E, 2.2 mg Vit. K,3.3 mg Riboflavin, 13.2 mg Pantothenic acid, 17.6 mg Niacin, 110.1 mgCholine, 1.98 ug B-12, 37.4 mg Mn, 0.6 mg I, 10 mg Cu, 0.3 mg Se, 100 mgZn, and 100 mg Fe per Kg of diet

Then, each pen received one of the four treatment diets. The positivecontrol group (+C) received the basal diet supplemented with dicalciumphosphate. The negative control group (−C) received just the basal diet.The yeast phytase group (YP) received the basal diet supplemented withthe expressed E. coli phytase at 1,200 U/kg of feed. The microbialphytase group (MP) received the basal diet supplemented with the BASFphytase at 1,200 U/kg of feed. Pigs were given free access to feed andwater. Body weight gain of individual pigs was recorded weekly. Dailyfeed intake of individual pens was recorded daily. Blood samples fromeach of the individual pigs were taken weekly to assay plasma inorganicphosphorus concentrations. The results of body weight (BW), averagedaily gain (ADG), average feed intake (ADFI), and feed/gain ratio (F:G),and plasma inorganic phosphorus (PP) are presented in Table 14.

TABLE 14 Summary of PP, BW, ADG, ADFI, and F:G of Pigs as Effected byDietary Phytase.¹ +C −C YP MP Initial PP 12.99 13.02 13.07 13.54 BW11.54 11.63 12 11.5 Week 1 PP 10.83^(A)  6.48^(C)  8.59^(B)  8.35^(B) BW14 13.83 14.29 13.92 ADG  .351  .316  .327  .345 ADFI  .700  .684  .697 .697 F:G  2.04  2.20  2.18  2.13 Week 2 PP  9.76^(A)  5.64^(D) 8.72^(B)  7.84^(C) BW 18.04 17.42 17.83 17.71 ADG  .578  .512  .506 .542 ADFI  .833  .855  .784  .837 F:G  1.46^(B)  1.67^(A)  1.56^(AB) 1.55^(AB) Week 3 PP 11^(A)  6.26^(C)  8.64^(B)  8.13^(B) BW 22.58 21.1722 22.21 ADG  .649^(A)  .536^(B)  .595^(AB)  .643^(AB) ADFI  1.166  1.02 1.001  1.003 F:G  1.8  1.92  1.71  1.36 Week 4 PP 10.94^(A)  6.31^(C) 9.65^(B)  9.2^(B) BW 27.54 25.29 27.79 27.38 ADG  .708^(AB)  .589^(B) .827^(A)  .738^(AB) ADFI  1.395^(A)  1.049^(B)  1.309^(A)  1.273^(AB)F:G  1.98  1.87  1.59  1.73 ¹Numbers in the same row without sharing acommon letter are significantly different. Analysis of difference wasconducted with the Bonferroni (Dunn) T-tests with alpha = 0.05 and df =20

In addition, there was severe phosphorus deficiency in the negativecontrol group in the end of the four-week experiment. But, there was nosign of phosphorus deficiency in the other three groups. Clearly, theexpressed E. coli phytase by Pichia was at least, if not more, effectiveas the commercial microbial phytase in improving bioavailability ofphytate-phosphorus from the corn-soybean meal diets for weanling pigs.It can be used to replace inorganic phosphorus supplementation toweanling pigs.

Example 20 Effects of the Expressed E. coli AppA Phytase by Pichiapastoris on Iron (Fe) and Phytate Phosphorus Bioavailability to WeanlingPigs

To determine the effect of the overexpressed E. coli phytase by Pichiaon dietary phytate-bound Fe bioavailability to weanling pigs, 20 anemicpigs (21 days old and 7.3 g hemoglobin (Hb)/dL blood) were selected. Thepigs were fed an Fe-deficient creep feed for 7 days and housed inmetabolic cages at the age of 28 days old. The pigs were then fed theexperimental diets at the age of 35 days old for 5 weeks. The treatmentdiets were as follows: Fe-deficient basal diet (−C, with added inorganicphosphorus), Fe-supplemented diet (+C), the Fe- and phosphorus-deficientdiet supplemented with the expressed E. coli phytase (YP), or thecommercial microbial phytase (BASF, MP) at 1,200 U/kg of feed. Bodyweight (BW), packed cell volume (PCV), Hb, and plasma inorganicphosphorus (PP) were determined weekly. The results are presented inTable 15.

TABLE 15 Summary of PCV, Hb, BW, and PP of Pigs as Effected by DietaryPhytase.¹ +C −C YP MP Initial PCV 25 25 26 24 Hb  7.73  7.22  7.85  7.08BW  8.14  8.27  8.17  7.45 PP  7.92  7.76  7.21  7.36 Week 1 PCV 25 2629 27 Hb  7.62  8.3  8.77  7.88 BW  9.44  8.84  9.63  8.57 PP  8.41 8.45  8.48  8.22 Week 2 PCV 29 26 30 28 Hb  8.6  7.34  8.93  8.27 BW12.32 10.13 11.91 10.84 PP 10.28^(a)  9.05^(ab)  8.89^(ab)  8.22^(a)Week 3 PCV 36^(a) 29^(b) 34^(a) 33^(a) Hb 11.55^(a)  8.2^(b) 10.84^(a) 9.96^(ab) BW 16.77^(a) 13^(b) 15.62^(ab) 14.62^(ab) PP 12.14^(a)11.37^(ab) 10.25^(bc)  9.71^(c) Week 4 PCV 39 34 38 36 Hb 12.99^(a)10.11^(b) 12.27^(a) 11.35^(ab) BW 21.36^(a) 17.37^(b) 19.44^(ab)18.56^(ab) PP 10.19^(a)  9.34^(ab)  9.49^(ab)  8.8^(b) Week 5 PCV 40 3840 39 Hb 13.52^(a) 12.24^(b) 13.64^(a) 13.13^(ab) BW 26.53 22.59 24.2723.43 PP  9.27^(a)  8.95^(ab)  8.79^(ab)  8.02^(n) ¹Values are means (n= 5). Means within the same row without sharing a common superscriptltter are significantly different (P < 0.10).

In conclusion, the overexpressed E. coli phytase by Pichia was at leastas effective as the BASF phytase in improving phytate-phosphorus and Feutilization in corn-soy diets for weanling pigs.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and thesetherefore are considered within the scope of the invention as defined inthe claims which follow.

1. A method of producing phytase in yeast, the method comprising:providing a polynucleotide encoding a phytase from Escherichia coli;expressing the polynucleotide in a yeast; and isolating the expressedprotein or polypeptide, wherein said protein or polypeptide catalyzesthe release of phosphate from phytate.
 2. The method of claim 1 whereinsaid polynucleotide encodes E. coli AppA phytase.
 3. The method of claim1 wherein said yeast is a Pichia species.
 4. The method of claim 1wherein said polynucleotide is carried on a nucleic acid vector.